instmctions): Our overall objective is to develop approaches for effective vaccination against pulmonary tularemia. During the previous funding period, we made considerable progress, including the demonstration that significant protection in C57BL/6 mice against the highly virulent type A strain of F. tularensis {Ft), SchuS4, can be induced by i.n. inoculation of FcR-targeted inactivated LVS {\Ft). This is the first case to our knowledge in wliich an inactivated vaccine has provided protection against Ft SchuS4. In this renewal application, we will further optimize conditions for mucosal vaccination, characterize the effects of pulmonary vaccination, and define the cellular and humoral mechanisms responsible for protective immunity against the highly virulent type A strain, SchuS4. Specifically, we will: 1) Determine the ability of i.n. vaccination with FcR-targeted immunogens, in the absence or presence of exogenous IL-12, to enhance the immune response to, and levels of protection against, mucosal (i.n.) challenge with Ft SchuS4. Two separate approaches will be used to target \Ft to FcR: a) administration of preformed mAb-IFf complexes, and b) simultaneous inoculation of uncomplexed \Ft plus anti-Ff mAb; 2) Establish the mechanisms responsible for enhanced induction of immunity by assessing the distribution of FcR targeted \Ft to tissues and lymphoid organs to determine if enhanced localization of '\Ft to secondary lymphoid tissues, and APC within these tissues, occurs as a result of FcR targeting. It will also be determined if enhanced \Ft processing and presentation results from targeting to FcR on APC; and 3) Establish the effector mechanisms responsible for enhanced protection after i.n. vaccination with FcR-targeted bacteria. The mechanisms responsible for protection after mucosal vaccination will be investigated by passive transfer of anti-F/ antibody or cells to naive mice with particular attention paid to examining a potential requirement for synergy between humoral and cellular immune mechanisms for induction of effective protection. The roles of TLR/NLRs and ROS/RNS in both inductive and effector phases will be examined by using genetically deficient mice and specific agonists/antagonists, in consultation with the PLs of subprojects 2 and 3. The results of subproject 1 will allow the design of new mucosal vaccination strategies for effective biodefense against infection with virulent Ft and will provide novel insight into the pulmonary immune mechanisms that are responsible for protection against respiratory tularemia. RELEVANCE (See Instruct'ons): The results of this subproject will allow the design of new mucosal vaccination strategies for effective biodefense against infection with virulent Ft and will provide novel insight into the pulmonary immune mechanisms that are responsible for protection against respiratory tularemia.